The goal of this proposal is to isolate and characterize melanoma tumor stem cells (MTSCs) from the patients diagnosed with various stages of this disease. Tumor stem cells (TSCs) are often insensitive to growth arresting signals and have infinite proliferative capacity allowing them to re-establish the entire tumor cell population eliminated after traditional medical regiments even with more aggressive phenotype. Thus, to treat cancer efficiently TSCs have to be identified and their properties analyzed to design the therapies that specifically target and eradicate all of them. Specifically, our studies will address the existence of MTSCs at each stage of melanoma development and analyze the role of stem cell self-renewing pathways in maintaining tumorigenic potential of MTSCs. We will achieve our goal by completing following specific aims: 1) To identify and purify melanoma tumor stem cells (MTSCs) from human melanoma samples;2) To analyze MTSCs for activation of signaling pathways involved in self-renewal and maintenance of normal stem cells. Our research design is based on the advanced techniques for stem cell sorting and identification of stem cell related markers developed in our laboratory. We plan to pursue aim 1 in the following steps: i) to determine candidate cell surface markers suitable for enrichment of MTSCs using combinatorial mAb staining and fluorescent activated cell sorting (FACS);ii) to determine tumor stem cell identity of fractionated melanoma cells in-vitro by performing tumor sphere formation and proliferation assays;iii) to determine tumor stem celt identity and purity of candidate MTSCs in-vivo by performing tumor xenograft and serial transplantation assays in immunocompromised mice. To understand molecular mechanisms that might be involved in emergence and maintenance of MTSCs we will analyze these cells for activation of signaling pathways involved in self-renewal and maintenance of normal stem cells. This aim will be pursued by utilizing lentiviral reporter assays combined with RNAi mediated mechanism of gene suppression to asses the role of candidate transcription factors in tumorigenic potential of MTSCs. Our studies will contribute to the new understanding of mechanisms underlying the origins of melanoma and its progression. Completion of our aims will identify new cellular and molecular targets for drug and immune melanoma therapies. Isolated MTSCs can be transplanted into immunodeficient mice and such tumor bearing mice will become a very useful model for preclinical testing of new melanoma treatments and diagnostics. These procedures can be designed to specifically irradicatge all of MTSCs and thus improve patient survival rate and functional outcome of tumor therapy.